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. 2004 Feb 17;101(7):2046-51.
doi: 10.1073/pnas.0308347100. Epub 2004 Feb 5.

Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes

Moon-Il Kang  1 , Akira Kobayashi, Nobunao Wakabayashi, Sang-Geon Kim, Masayuki Yamamoto
Affiliations

Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes

Moon-Il Kang et al. Proc Natl Acad Sci U S A. .

Abstract

Transcription factor Nrf2 regulates basal and inducible expression of phase 2 proteins that protect animal cells against the toxic effects of electrophiles and oxidants. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a multidomain, cysteinerich protein that is bound to the actin cytoskeleton. Keap1 acts both as a repressor of the Nrf2 transactivation and as a sensor of phase 2 inducers. Electrophiles and oxidants disrupt the Keap1-Nrf2 complex, resulting in nuclear accumulation of Nrf2, where it enhances the transcription of phase 2 genes via a common upstream regulatory element, the antioxidant response element. Reporter cotransfection-transactivation analyses with a series of Keap1 deletion mutants revealed that in the absence of the double glycine repeat domain Keap1 does not bind to Nrf2. In addition, deletion of either the intervening region or the C-terminal region also abolished the ability of Keap1 to sequester Nrf2, indicating that all of these domains contribute to the repressor activity of Keap1. Immunocytochemical and immunoprecipitation analyses demonstrated that Keap1 associates with actin filaments in the cytoplasm through its double glycine repeat domain. Importantly, disruption of the actin cytoskeleton promotes nuclear entry of an Nrf2 reporter protein. The actin cytoskeleton therefore provides scaffolding that is essential for the function of Keap1, which is the sensor for oxidative and electrophilic stress.

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Figures

Fig. 1.
Fig. 1.
Keap1 colocalizes with actin filaments in the cytoplasm. (A) Schematic presentation of Keap1 based on Swiss-Prot, using the Sanger Center Database. We assigned five domains within Keap1: NTR, BTB, IVR, DGR, and CTR. (B–E) Cytoplasmic localization of Keap1 in NIH 3T3 cells. Keap1 was expressed in NIH 3T3 cells, and subcellular localization of Keap1 was detected immunohisto-chemically by two anti-Keap1 antibodies against the N- and C-terminal ends of Keap1 (B and C, respectively). Bright-field microscopic images for B and C are shown in D and E, respectively. (F–K) colocalization of Keap1 and actin filaments in MEF derived from transgenic mouse embryos expressing Keap1. Subcellular localization of actin filaments (F and I) and Keap1 (G and J) are visualized by staining with phalloidin conjugated with Texas red and anti-Keap1 antibody, respectively. H and K show merged signals. Fluorescence was recorded by confocal microscopy. (Scale bar, 40 μm.)
Fig. 2.
Fig. 2.
Keap1 interacts with actin filaments through the DGR domain. (A) Schematic presentation of the structure of Keap1 deletion mutants. (B) Immunoprecipitation with whole-cell extracts of 293T cells expressing deletion mutants of Keap1. Immunoprecipitates (IP) obtained by anti-actin antibody were subjected to immunoblot analysis (IB) with anti-Keap1 antibody (Upper). The expression level of Keap1 deletion mutants was verified by immunoblot analysis (Lower). Analysis with wild-type Keap1-transfected cell lysates (lanes 1 and 8) as well as cell lysates transfected with Keap1 mutant ΔNTR (lane 2), ΔBTB (lane 3), ΔIVR (lane 4), ΔCTR (lane 5), and ΔDGR (lane 7) are shown. Lane 6 is loaded with cell extract expressing Nrf2 but not Keap1. Two anti-Keap1 antibodies were used: one against CTR (lanes 1–6) and the other against NTR (lanes 7 and 8).
Fig. 3.
Fig. 3.
Disruption of actin filaments triggers nuclear transport of Neh2-GFP. (A) NIH 3T3 cells were stained with phalloidin conjugated with Texas red and 4′,6-diamidino-2-phenylindole (DAPI) after addition of DMSO (A; a vehicle control) or cytoskeletal filament disruptors, cytochalasin B (B), swinholide A (C), or colchicine (D). (E) subcellular localization of Neh2-GFP and Keap1 after treatment with cytochalasin B. Cells (4 × 103) were transfected with expression plasmids of Keap1 (0.2 μg) and Neh2-GFP, a reporter protein of Nrf2 (0.8 μg). The latter is a fusion protein of Neh2 domain and GFP. Localization of these proteins was examined by fluorescence microscopy with use of GFP fluorescence and anti-Keap1 antibody, respectively (first and second rows). Merged images of Neh2-GFP and Keap1 signals are shown in the third row. Nuclei are shown with DAPI staining (fourth row). (Original magnification, ×400.) (F) nuclear transport of Neh2-GFP 3 h after the addition of cytochalasin B (Cyto-B), swinholide A (Swin-A), and colchicine (Col). Shown is the percentage of cells expressing Neh2-GFP in nucleus among the total transfected cells. The average and standard errors represent three independent transfection experiments.
Fig. 4.
Fig. 4.
CTR contributes to Keap1 activity retaining Neh2-GFP in cytoplasm. Subcellular localization of Neh2-GFP was examined in the presence of Keap1 deletion mutants. Transfection was performed as described in the legend to Fig. 3. Localization of Neh2-GFP and Keap1 mutant proteins was examined by fluorescence microscopy (first and second rows). Merged signals of both Neh2-GFP and Keap1 are shown in the third row. Nuclei are shown with DAPI staining (shown as Nucleus; fourth row). (Original magnification, ×400.)
Fig. 5.
Fig. 5.
IVR and CTR are both essential for Keap1 repression of Nrf2. (A) DGR of Keap1 directly associates with Nrf2. Whole-cell extracts prepared from 293T cells cotransfected with expression plasmids of various Keap1 deletion mutants (2 μg) and Flag-tagged Nrf2 (2 μg) were subjected to immunoprecipitation (IP). Immunoprecipitates obtained by anti-Flag antibody were subjected to immunoblot analysis (IB) with anti-Keap1 antibodies (Upper). The expression level of Keap1 deletion mutants was verified by immunoblot analysis (Lower). Analysis of cell lysates cotransfected with Nrf2 and wild-type Keap1 (lanes 3 and 9) as well as cell lysates cotransfected with Nrf2 and Keap1 ΔNTR (lane 4), ΔBTB (lane 5), ΔIVR (lane 6), ΔCTR (lane 7), or ΔDGR (lane 8) mutants are shown. Lane 1 is loaded with cell extract expressing only Keap1, and lane 2 is loaded with Nrf2 only. Anti-Keap1 CTR antibody was used in lanes 1–7, and anti-Keap1 NTR antibody was used for lanes 8 and 9. (B) Expression level of Nrf2 in immunoprecipitates was monitored by immunoblot analysis with anti-Nrf2 antibody. Analysis of cell lysates cotransfected with Nrf2 and wild-type Keap1 (lane 3) as well as cell lysates cotransfected with Nrf2 and Keap1 ΔNTR (lane 4), ΔBTB (lane 5), ΔIVR (lane 6), ΔCTR (lane 7), or ΔDGR (lane 8) mutants are shown. Lane 1 is loaded with cell extract expressing only Keap1, and lane 2 is loaded with Nrf2 only. (C) Three domains (DGR, CTR, and IVR) are crucial for the Keap1 activity. Expression plasmids of Nrf2 (90 ng) and various Keap1 deletion mutants (shown in the figure; 10 ng) were transfected into NIH 3T3 cells (2 × 104) along with a reporter plasmid, pNQO1-ARE (50 ng). Assays were performed in triplicate.
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